Interaction of the SHD domain of Mlph with Rab27a WT and mutant proteins.
pFlag-CMV4-Rab27a-WT or -mutants and pMyc-cDNA3.1-SHD-Mlph were cotransfected into 293T cells and their association was analyzed by immunoprecipitation with anti-Myc antibody one day following transfection. (A) Coimmunoprecipitated Flag-Rab27a-WT or-mutant detected by anti-Flag tag antibody (IP: anti-Myc; Blot: anti-Flag). The size difference observed between WT and mutant Rab27a proteins results from the different restriction sites used for their respective cloning. The same blot was then stripped and reprobed with anti-Myc antibody to ensure that the same amounts of pMyc-SHD-Mlph proteins had been loaded (IP: anti-Myc; Blot: anti-Myc). The top panel indicates the total expressed Flag-Rab27a (1:100 volume of the reaction mixture) used for immunoprecipitation. The positions of the markers (× 10−3) are shown on the left. (B) Coimmunoprecipitated pMyc-SHD-Mlph detected by anti-Myc tag antibody (IP: anti-Flag; Blot: anti-Myc). The same blot was then stripped and reprobed with anti-Flag antibody to ensure that the same amounts of Flag-Rab27a-WT or -mutant proteins had been loaded (IP: anti-Flag; Blot: anti-Flag). The top panel indicates the total expressed pMyc-SHD-Mlph (1:100 volume of the reaction mixture) used for immunoprecipitation.
